Porphobilinogen deaminase, (also known as porphobilinogen ammonia-lyase (polymerizing)), E.C. 4.3.1.8. (Waldenström 1937, J. Acta. Med. Scand. Suppl. 8) is the third enzyme in the heme biosynthetic pathway. In the following, this enzyme and the recombinant human form will be termed “PBGD” and “rhPBGD”, respectively.
PBGD is important in relation to Acute intermittent porphyria (AlP), which is an autosomal dominant disorder in man caused by a defect (50% reduction of activity) of PBGD (see WO01/07065 for further details in relation to this).
Smythe et al. (Biochem. J. (1988) 251:237–241) describes a purification protocol for PBGD. The volume of the PBGD containing extract loaded on the specified chromatography columns is less than 1 L (see table 1). In the present context this is considered a small-scale laboratory purification protocol. The described protocol comprises first loading the PBGD containing extract on a Ion-exchange column (DEAE-cellulose) followed by use of an affinity column (Cibacron Blue FG3-A-Sepharose).
In relation to an upscaled purification protocol for rhPBGD, WO01/07065 describes in example 7 a high scale fermentation process using a recombinant E.coli cell capable of expressing the rhPBGD followed by a down-stream purification process. The purification process comprises loading a rhPBGD containing extract, obtained from the fermentation, on Hydrophobic interaction chromatography (HIC) column followed by a Ion-exchange chromatography CIEC) step, ending with an affinity step on Cibacron Blue FF Sepharose. The used column volumes are around 10 to 12 L.